Inhibition of poly(A) polymerase requires p34cdc2/cyclin B phosphorylation of multiple consensus and non-consensus sites.
We showed previously that p34(cdc2)/cyclin B (MPF) hyperphosphorylates poly(A) polymerase (PAP) during M-phase of the cell cycle, causing repression of its enzymatic activity. Mutation of three cyclin-dependent kinase (cdk) consensus sites in the PAP C-terminal regulatory domain prevented complete phosphorylation and MPF-mediated repression. Here we show that PAP also contains four nearby non-consensus cdk sites that are phosphorylated by MPF. Remarkably, full phosphorylation of all these cdk sites was required for repression of PAP activity, and partial phosphorylation had no detectable effect. The consensus sites were phosphorylated in vitro at a 10-fold lower concentration of MPF than the non-consensus sites. Consistent with this, during meiotic maturation of Xenopus oocytes, consensus sites were phosphorylated prior to the non-consensus sites at metaphase of meiosis I, and remained so throughout maturation, while the non-consensus sites did not become fully phosphorylated until after 12 h of metaphase II arrest. We propose that PAP's multiple cdk sites, and their differential sensitivity to MPF, provide a mechanism to link repression specifically to late M-phase. We discuss the possibility that this reflects a general means to control the timing of cdk-dependent regulatory events during the cell cycle.
Colgan
D F
DF
Department of Biological Sciences, Columbia University, New York 10027, USA.
Murthy
K G
KG
Zhao
W
W
Prives
C
C
Manley
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002417:Cattle; D002454:Cell Differentiation; D016384:Consensus Sequence; D019926:Cyclin B / Q000378:metabolism*; D006801:Humans; D016200:Maturation-Promoting Factor / Q000502:physiology; D008540:Meiosis; D000090204:Mesothelin; D051379:Mice; D008938:Mitosis; D008969:Molecular Sequence Data; D009865:Oocytes / Q000166:cytology; D010766:Phosphorylation; D011062:Polynucleotide Adenylyltransferase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
D019926:Cyclin B; C000715528:Msln protein, mouse; D016203:CDC2 Protein Kinase; D016200:Maturation-Promoting Factor; D011062:Polynucleotide Adenylyltransferase; D000090204:Mesothelin